Under normal conditions, most T lymphocytes are made in the thymus. Unfortunately, the size of the thymus degenerates progressively with age, with a noticeable onset at around the time of puberty. Because the output of new T cells from the thymus is directly proportional to its mass, age-related thymic atrophy results in a lifelong, progressive decline in the production of new, naive T cells. In the peripheral lymphoid system, this decreased thymic output is compensated by homeostatic expansion of existing T cells. While this avoids frank T cell lymphopenia, the end result is that, over time, the T cell pool increasingly represents an oligoclonal repertoire, rather than the broad, unbiased spectrum of immunity conferred by newly generated, naive thymic T cells. Thus, aging is associated with an accumulation of T cell immunodeficiencies (or immunoinsufficiencies) that, in turn, result in increased susceptibility to infectious disease, decreased response to vaccines, decreased anti-tumor surveillance, increased autoimmunity, and other related disorders. Decreased capacity to make new T cells is also a substantial limitation in hematopoietic stem cell transplantation, which is an established therapy for diseases like leukemia, and an emerging therapy for autoimmune disorders like lupus erythematosus and multiple sclerosis. Correcting and/or preventing age-related thymic degeneration (atrophy) is thus of substantial importance for enhancing quality of life, and for decreasing health care costs in adults and the elderly. Notably, the thymus can be induced to completely regrow, although the most efficient means for this (surgical castration) is somewhat impractical. Nonetheless, this plasticity underscores the potential for devising other more practical means for inducing thymic regrowth. The aims of this project are to use recently devised, robust physical methods (laser microdissection, microarray) and computational modeling to generate accurate global lists of thymic stromal genes in their native state in situ, both in the unmodified atrophied thymus, and during various phases of regrowth (initiation, log phase, termination) induced by castration. Stromal gene expression signatures will then be analyzed to reveal changes that occur in atrophy, and during the regrowth response. Informatic and biological validations will be used to identify key regulators in these processes, which will be followed-up by conventional biological approaches (transgenics, knockouts). In addition to an in- depth understanding of thymic stromal biology and the regrowth process, this project is expected to ultimately reveal potential new targets for therapeutic approaches for thymic regeneration. PUBLIC HEALTH RELEVANCE: Decreases in T cell immunity are a direct consequence of age-related thymic degeneration, and are linked to a host of undesirable conditions, including increased risk of infection, increased severity of infection, decreased response to vaccinations, decreased tumor surveillance, increased autoimmunity, and so on. Regenerating the thymus is therefore of significant interest to human health. This application proposes a novel approach to understanding the biology of regrowth of the atrophied thymus. In addition to an in-depth understanding of the regrowth process, this project is expected to ultimately reveal potential new targets for thymic regeneration therapy.